COMPOSITION FOR INHIBITING OSTEOCLASTS CONTAINING AGASTACHE RUGOSA EXTRACT AS ACTIVE INGREDIENT, AND USE THEREOF

Abstract

The Agastache rugosa extract or the isoagastachoside or crude polysaccharide fraction included in the extract according to the present invention can be prepared based on plants being domestically cultivated, and thus is advantageous in terms of time and cost, and can effectively inhibit the differentiation, growth, and bone resorption functions of osteoclast progenitor cells, thus increasing bone density and the like, and accordingly, can be effectively used for the prevention, amelioration, or treatment of diseases related to bone loss caused by differentiation into osteoclasts or excessive activity of osteoclasts. In addition, the active ingredient, which is a plant-derived natural substance, is safe due to fewer side effects on the human body compared to chemical drugs, and inhibits osteoclast progenitor cells independently of estrogen, and thus can be widely used in osteoporosis occurring due to various causes other than menopause.

Claims

1. A health functional food composition for preventing or ameliorating a disease related to bone loss caused by osteoclasts, the composition containing an Agastache rugosa extract as an active ingredient.

2. The health functional food composition of claim 1, wherein the Agastache rugosa extract is isoagastachoside or crude polysaccharide fraction contained in the Agastache rugosa extract.

3. The health functional food composition of claim 1, wherein the Agastache rugosa extract is extracted from any one or more selected from the group consisting of leaves, stems, flowers and fruits of Agastache rugosa.

4. The health functional food composition of claim 1, wherein the Agastache rugosa extract is extracted using as a solvent any one or more selected from the group consisting of water, an organic solvent, and a mixture thereof.

5. The health functional food composition of claim 1, wherein the Agastache rugosa extract is extracted by any one or more extraction methods selected from the group consisting of reduced pressure high temperature extraction, boiling water extraction, reflux extraction, hot water extraction, cold extraction, room temperature extraction, ultrasonic extraction, or vapor extraction.

6. The health functional food composition of claim 1, wherein the molecular weight of the crude polysaccharide fraction is 1 to 300 kDa.

7. The health functional food composition of claim 1, wherein the composition inhibits the differentiation into osteoclasts or the activity of osteoclasts.

8. The health functional food composition of claim 1, wherein the disease related to bone loss is any one or more selected from the group consisting of osteoporosis, Pajet's disease of bone, rickets, osteomalacia, renal osteodystrophy in patients with renal failure, rheumatoid bone disease, degenerative bone disease, hone metastatic lesion, and a primary bone tumor.

9-12. (canceled)

13. A composition for inhibiting bone loss caused by osteoclasts, the composition containing an Agastache rugosa extract as an active ingredient.

14. The composition for inhibiting bone loss of claim 13, wherein the Agastache rugosa extract is isoagastachoside or crude polysaccharide fraction contained in the Agastache rugosa extract.

15. The composition for inhibiting bone loss of claim 13, wherein the composition inhibits the differentiation into osteoclasts or the activity of osteoclasts.

16. A composition for inhibiting the differentiation of osteoclast progenitor cells into osteoclasts, the composition containing an Agastache rugosa extract as an active ingredient.

17. The composition for inhibiting the differentiation of osteoclast progenitor cells into osteoclasts of claim 16, wherein the Agastache rugosa extract is isoagastachoside or crude polysaccharide fraction contained in the Agastache rugosa extract.

18. A method for preventing or treating diseases related to bone loss caused by osteoclasts, the method comprising administering an Agastache rugosa extract to a patient with a disease related to bone loss caused by osteoclasts.

19. The method for preventing or treating of claim 18, wherein the Agastache rugosa extract is isoagastachoside or crude polysaccharide fraction contained in the Agastache rugosa extract.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The above and other aspects, features, and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing embodiments thereof in detail with reference to the accompanying drawings, in which:

[0076] FIG. 1 shows the component analysis results of a hot water extract of Agastache rugosa (WEAR). It was confirmed that Agastache rugosa's WEAR contained isoagastachoside component (0.2% of the weight of the WEAR).

[0077] FIG. 2 shows the number of osteoclasts according to the treatment concentration when the bone marrow cell-derived macrophages are treated with a hot water extract of Agastache rugosa (WEAR, a)) or an ethanol extract of Agastache rugosa (EEAR, b)). It was confirmed that both WEAR and EEAR had the effect of significantly inhibiting the differentiation into osteoclasts.

[0078] FIG. 3 shows the number of osteoclasts according to the treatment concentration when the bone marrow cell-derived macrophages are treated with a hot water extract of Agastache rugosa (WEAR), an isoagastachoside or a polysaccharide fraction (WEAR-PS) separated from WEAR of Agastache rugosa. WEAR also had the effect of inhibiting the differentiation of macrophages into osteoclasts, and isoagastachoside or WEAR-PS more significantly inhibited the differentiation into osteoclasts.

[0079] FIG. 4 shows the number of osteoclasts according to the treatment concentration, when ethanol precipitation is performed on the hot water extract of Agastache rugosa (WEAR) and the bone marrow cell-derived macrophages are treated with the precipitated crude polysaccharide fraction (WEAR-PO), the non-precipitated fraction (WEAR-ED) or crude polysaccharide fractions obtained by separating WEAR-PO by molecular weight cut-off (P0-3 kDA Fx, P0-10 kDA Fx, P0-30 kDA Fx, P0-100 kDA Fx, P0-300 kDA Fx, P0-1000 kDA Fx). WEAR-ED, P0-300 kDA Fx or P0-1000 kDA Fx fractions were confirmed to have insignificant inhibitory effect on osteoclast differentiation.

[0080] FIG. 5 shows the viability of bone marrow cell-derived macrophages (progenitor cells of osteoclasts) according to the treatment concentration when the bone marrow cell-derived macrophages are treated with a hot water extract of Agastache rugosa (WEAR, a)), an ethanol extract of Agastache rugosa (EEAR, b)) or isoagastachoside (c)). WEAR or isoagastachoside did not affect the viability of osteoclast progenitor cells, and it was confirmed that EEAR had an effect of inhibiting the survival of osteoclast progenitor cells when it was 100 μg/ml or more.

[0081] FIG. 6 shows an image of mineral pit resorbed by osteoclasts and a resorption area according to the treatment concentration of the hot water extract of Agastache rugosa (WEAR). It was confirmed that as the concentration of the treated WEAR increased, the bone resorption capacity of osteoclasts was inhibited and the area of the mineral pit decreased.

[0082] FIG. 7 shows the differentiation of osteoblasts according to the treatment concentration of hot water extract of Agastache rugosa (WEAR). It was confirmed that WEAR did not affect the differentiation of osteoblasts.

[0083] FIG. 8 shows changes in body weight after oral administration of hot water extract of Agastache rugosa (WEAR) to an ovariectomized (OVX) mouse model. It was confirmed that WEAR had the effect of inhibiting the weight change in the ovariectomized mouse model.

[0084] FIG. 9 shows changes in uterine weight after oral administration of hot water extract of Agastache rugosa (WEAR) to an ovariectomized (OVX) mouse model. It was confirmed that WEAR did not affect the uterine weight in the ovariectomized mouse model.

[0085] FIG. 10 respectively shows computed tomography 3D images of trabecular bone of the thigh bone (a)), changes in bone volume (BV/TV, b)), changes in trabecular bone thickness (Tb.Th, c)), and changes in trabecular bone count (Tb.N, d)) after oral administration of hot water extract of Agastache rugosa (WEAR) to an ovariectomized (OVX) mouse model. It was confirmed that WEAR had the effect of suppressing the decrease in bone volume, trabecular bone thickness, and trabecular bone number in the ovariectomized mouse model.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1

[0086] Preparation of Agastache rugosa Extract

[0087] <1-1> Preparation of Agastache rugosa Extract

[0088] For hot water extract of Agastache rugosa (WEAR), 500 g of Agastache rugosa added to 3.5 L of distilled water, extracted under reflux for 3 hours (heating mantle), filtered through a qualitative filter paper (Φ185 mm), and freeze-dried. In cell experiments, the hot water extract of Agastache rugosa (WEAR) dissolved in distilled water was filtered through a 0.22 filter and then used for the experiment. In animal experiments, the freeze-dried extract was dissolved in sterile water for injection and used.

[0089] For ethanol extract of Agastache rugosa (EEAR), 500 g of Agastache rugosa was added to 3.5 L of 70% ethanol, extracted under reflux (heating mantle) for 3 hours, filtered through a qualitative filter paper (Φ185 mm), concentrated with a rotary evaporator, and then freeze-dried. After dissolving in dimethyl sulfoxide, ethanol extract of Agastache rugosa (EEAR) was used for the experiment.

[0090] <1-2> Preparation of (Crude) Polysaccharide Fraction

[0091] To prepare a polysaccharide fraction from the hot water extract of Agastache rugosa (WEAR) prepared in Example <1-1>, 4 times the volume of ethanol was added to the WEAR and precipitated with 80% ethanol. The precipitated polysaccharide was recovered and polysaccharide fractions having a molecular weight of 10 kDa or more were obtained using a permeable membrane (Amicon Ultra-15 10 k). The obtained fractions were dried using a Genevac EZ-2 plus Evaporating system, and the polysaccharide fractions (WEAR-PS) dissolved in distilled water were filtered through a 0.22 filter and then used for the experiment.

[0092] Additionally, when performing 80% ethanol precipitation for WEAR, a precipitated crude polysaccharide fraction (WEAR-PO) and a non-precipitated fraction (WEAR-ED) are obtained, freeze-dried, dissolved in distilled water, filtered with a 0.22 filter, and then used for the experiment. In addition, the WEAR-PO was passed through a filtration membrane having a molecular weight cut-off of 3, 10, 30, 100, 300 and 1000 kDa to obtain a crude polysaccharide fraction that did not pass (P0-3 kDA Fx, P0-10 kDA Fx, P0-30 kDA Fx, P0-100 kDA Fx, P0-300 kDA Fx, P0-1000 kDA Fx), freeze-dried, dissolved in distilled water, filtered through a 0.22 , and then used for the experiment.

Example 2

[0093] Component Analysis of Agastache rugosa Extract

[0094] We tried to determine whether isoagastachoside is included in the Agastache rugosa extract, especially the hot water extract of Agastache rugosa (WEAR).

[0095] Specifically, it was analyzed under gradient elution conditions by using UHPLC-MS/MS (Dionex UltiMate 3000 UHPLC and Thermo Q-Exactive mass spectrometer); as a stationary phase, a column of Acquity BEH C18 column (100×2.1 mm, 1.7 μm); and as a mobile phase, water with 0.1% formic acid added and acetonitrile. The mass spectrometer used HESI source and analyzed in negative ion mode. Retention time, mass spectrum, m/z, and fragment were compared with isoagastachoside standard product (99%, Ensol Bio).

[0096] As a result, as shown in FIG. 1 a), a peak of isoagastachoside was observed at a retention time of 12.36 minutes on the chromatogram of the standard product and the chromatogram of WEAR. In addition, as shown in FIG. 1 b), it was confirmed that isoagastachoside was contained in the hot water extract by confirming the [M+CHO.sub.2]— form precursor ion (MS1, m/z 533.1304) and the [M—C.sub.8H.sub.13O.sub.6] form product ion (MS2, m/z 285.0613) on the mass spectrum of the observed peak. As a result of the quantitative test, it was confirmed that isoagastachoside was contained in the WEAR in an amount of 0.2% by weight.

Example 3

[0097] Analysis of the Effect of Agastache rugosa Extract on Osteoclast Differentiation

[0098] <3-1> Inhibitory Effect of Agastache rugosa Extract on Osteoclast Differentiation

[0099] We tried to determine whether Agastache rugosa extract inhibits the differentiation of osteoclast progenitor cells into osteoclasts. The osteoclast progenitor cells were treated with hot water extract of Agastache rugosa (WEAR) and ethanol extract of Agastache rugosa (EEAR) by concentration, and the number of osteoclasts was analyzed accordingly.

[0100] Specifically, bone marrow cells of mice were cultured in α-MEM medium containing M-CSF (60 ng/ml) and 10% FBS for 3 days to obtain bone marrow cell-derived macrophages (BMMs), which were used as osteoclast progenitor cells. To generate osteoclasts, 1×10.sup.4 pieces of the obtained BMMs were placed in per well of a 96-well plate, cultured for 4 days in a medium containing M-CSF (60 ng/ml) and RANKL (50 ng/ml), and the BMM were treated with hot water extract of Agastache rugosa (WEAR; 10, 33, 100, 200 μg/ml) or ethanol extract of Agastache rugosa (EEAR; 10, 33, 100 μg/ml) under the same culture condition. After fixing the cells with 10% formalin, TRAP (Tartrate-resistant acid phosphatase) staining was performed using naphthol AS-MS phosphate and Fast red violet LB salt, and TRAP-positive cells having three or more nuclei (osteoclasts) were counted. Data are presented as mean±standard deviation, and statistical analysis was performed using one-way ANOVA with Dunnett test, **p<0.01.

[0101] As a result, as shown in FIG. 2 a), the osteoclast differentiation inhibitory effect of 35, 77, and 98% was exhibited by treatment with 33, 100, and 200 μg/ml of hot water extract of Agastache rugosa (WEAR), respectively. In addition, as shown in FIG. 2 b), the osteoclast differentiation inhibitory effect of 27% and 91% was exhibited by treatment with 33 and 100 μg/ml of ethanol extract of Agastache rugosa (EEAR), respectively.

[0102] <3-2> Inhibitory Effect of Components Contained in Agastache rugosa Extract on Osteoclast Differentiation

[0103] We tried to determine whether isoagastachoside or polysaccharide fraction contained in the extract of Agastache rugosa inhibits the differentiation of osteoclast progenitor cells into osteoclasts.

[0104] Specifically, in the same manner as in Example <3-1>, it was assessed whether hot water extract of Agastache rugosa (WEAR; 5, 10 and 20 μg/ml), isoagastachoside (5 and 10 μg/ml), or the polysaccharide fraction (WEAR-PS; 3.3, 10, 33, 100, 200 μg/ml) separated from WEAR in Example <1-2> had an inhibitory effect on osteoclast differentiation. IC50 (inhibitory concentration 50), the concentration showing 50% inhibition efficiency, was calculated using PraphPad Prism software.

[0105] As a result, as shown in FIG. 3 a), it was confirmed that isoagastachoside contained in hot water extract of Agastache rugosa (WEAR) had the effect of inhibiting the differentiation of bone marrow cell-derived macrophages (BMM) into osteoclasts. Isoagastachoside showed a better inhibitory effect on osteoclast differentiation than WEAR at the same concentration, particularly when treated with 10 μg/ml, inhibited osteoclast differentiation very significantly compared to WEAR at the same concentration. This meant that isoagastachoside acted as an active ingredient of WEAR's osteoclast differentiation inhibitory effect.

[0106] In addition, as shown in FIG. 3 b), the WEAR osteoclast differentiation inhibitory 1050 value was 66.84 μg/ml, whereas the WEAR-PS 1050 value was 16.97 μg/ml, indicating that the inhibitory effect of WEAR-PS on osteoclast differentiation was about 4 times more effective. This meant that WEAR-PS acted as an active ingredient of WEAR's osteoclast differentiation inhibitory effect.

[0107] <3-3> Analysis of (Crude) Polysaccharide Fractions Contained in Agastache rugosa Extract

[0108] In Example <3-2>, since it was confirmed that the polysaccharide fraction contained in the hot water extract of Agastache rugosa (WEAR) had an osteoclast differentiation inhibitory effect, we specifically wanted to confirm the molecular weight of the polysaccharide fraction exhibiting the osteoclast differentiation inhibitory effect.

[0109] Specifically, WEAR-PO, WEAR-ED, P0-3 kDA Fx, P0-10 kDA Fx, P0-30 kDA Fx, P0-100 kDA Fx, P0-300 kDA Fx and P0-1000 kDA Fx separated in Example <1-2> and WEAR were treated at concentrations of 10, 33, 100, and 200 μg/ml, respectively, and the osteoclast differentiation inhibitory effect was assessed in the same manner as in Example <3-1>.

[0110] As a result, as shown in FIG. 4, it was confirmed that WEAR-PO had a superior osteoclast differentiation inhibitory effect compared to WEAR, and WEAR-ED had a significantly reduced osteoclast differentiation inhibitory effect compared to WEAR or WEAR-PO. In addition, among the crude polysaccharide fractions of WEAR, P0-3 kDA Fx, P0-10 kDA Fx, P0-30 kDA Fx and P0-100 kDA Fx showed excellent osteoclast differentiation inhibitory effects, especially the effects of P0-10 kDA Fx, P0-30 kDA Fx and P0-100 kDA Fx were significantly superior. On the other hand, P0-300 kDA Fx and P0-1000 kDA Fx did not show osteoclast differentiation inhibitory effect.

Example 4

[0111] Analysis of the Effect of Agastache rugosa Extract on Cell Survival

[0112] We tried to determine whether Agastache rugosa extract or isoagastachoside contained therein inhibits the survival of osteoclast progenitor cells.

[0113] Specifically, 1.2×10.sup.4 pieces of bone marrow cell-derived macrophages (BMMs) were placed in per well of a 96-well plate, and then cultured for 2 days in a medium containing M-CSF(60 ng/) in which hot water extract of Agastache rugosa extract (WEAR; 5, 10, 20, 33, 100 and 200 μg/ml), ethanol extract of Agastache rugosa (EEAR; 10, 33 and 100 μg/ml) or isoagastachoside (5 and 10 μg/ml) samples were added. Cell viability was determined by measuring absorbance at 450 nm using Cell counting Kit-8 (CCK-8, Dojindo Inc.), percentage of untreated controls and data are presented as mean±standard deviation, and statistical analysis was performed using one-way ANOVA with Dunnett test, **p<0.01.

[0114] As a result, there was no effect in cell survival when treated with WEAR or isoagastachoside (a) and c) of FIG. 5). On the other hand, in the case of Agastache rugosa ethanol extract (EEAR), there was no difference in cell survival even when treated with concentrations of 10 and 33 μg/ml, but cell survival was reduced by 23% when treated with concentrations of 100 μg/ml (FIG. 5 b)).

Example 5

[0115] Analysis of the Effect of Agastache rugosa Hot Water Extract on Osteoclast Bone Resorption Capacity

[0116] We tried to determine the effect of Agastache rugosa hot water extract (WEAR) on the bone resorption function of osteoclasts.

[0117] Specifically, 2×10.sup.4 pieces of bone marrow cell-derived macrophages (BMMs) of Example 4 were placed in per well of a mineral-coated 96-well plate (Osteo Assy Surface, corning, NY, USA), and then were cultured for 4 days in a medium containing M-CSF (60 ng/ml) and RANKL (100 ng/ml). In the same culture conditions, BMMs were treated with the concentrations (33, 100, 200 μg/ml) of hot water extract of Agastache rugosa (WEAR). The mineral pit (resorption pit) resorbed by the osteoclasts was expressed as mean±standard deviation by removing the osteoclasts using clorox, then taking a photomicrograph and analyzing the resorption area using image J software, and statistical analysis was performed using one-way ANOVA with Dunnett test, *p<0.05 and **p<0.01.

[0118] As a result, as shown in FIG. 6, when hot water extract of Agastache rugosa (WEAR) was treated at concentrations of 33, 100 and 200 μg/ml, compared to the control group, the bone resorption function exhibited 21, 30 and 67% inhibitory effects, respectively.

Example 6

[0119] Analysis of the Effect of Agastache rugosa Hot Water Extract on Osteoblasts

[0120] Mineralization according to the differentiation of osteoblasts was measured to determine whether Agastache rugosa hot water extract (WEAR) affects the differentiation of osteoblasts other than osteoclasts.

[0121] Specifically, 1.7×10.sup.4 pieces of D1 ORL UVA cells (ATCC, USA), which are mouse bone marrow stromal cells, were placed in per well of a 96-well plate and then cultured for 2 days in a DMEM medium containing 10% FBS. Then, ascorbic acid (50 μg/ml), beta-glycerophosphate (5 mM) and Agastache rugosa extract hot water extract (WEAR; 0, 10, 33, 100 and 200 μg/ml) were added to an α-MEM medium containing 10% FBS and cultured for 8 days. Mineralization according to osteoblast differentiation after cell fixation was identified by Alizarin Res S staining, and for comparison of mineralization, the absorbance was measured after extraction of the stained Alizarin Red S with 10% acetic acid.

[0122] As a result, as shown in FIG. 7, it was confirmed that WEAR did not affect the differentiation of osteoblasts.

Example 7

[0123] Analysis of the Effect of Agastache rugosa Hot Water Extract on an Ovariectomized Mouse Model

[0124] To determine whether the bone loss-related effects of Agastache rugosa extract (WEAR) were related to estrogen, body weight, uterine weight, bone volume, trabecular bone thickness, and trabecular bone count were measured in an ovariectomized mouse model treated with WEAR.

[0125] Specifically, 30 6-week-old female C57BL/6J mice (Central Experimental Animal, Seoul) were acclimatized for 1 week, and bilateral ovariectomy (OVX, 30 mice) and sham surgery (Sham, 10 mice) were performed on the 7-week-old mice. After 1 week, feed (Saron Bio, D12492) containing 60% kcal fat was freely ingested, and 0.2 g (5 mice) and 0.6 g (5 mice) of Agastache rugosa hot water extract (WEAR) dissolved in sterile distilled water per kg of mouse was orally administered once daily for 4 weeks. Sterile distilled water was orally administered once daily for 4 weeks to the sham surgery group (Sham, 10 mice) and bilateral ovariectomy control group (10 mice). Changes in body weight of mice between 1 week and 5 weeks after OVX were measured, and the weight after hysterectomy through autopsy after 5 weeks of OVX were measured. In addition, after autopsy of the mouse, the thigh bone was removed and fixed with 10% neutral formalin. After micro-computed tomography (Quantum GX, Perkin Elmer, USA) of the thigh bone, the amount and structure of trabecular bone were analyzed (DataViewer software version 1.4.3.2, SkyScan). Data are presented as mean±standard error, and statistical analysis was performed using one-way ANOVA with Dunnett test, **p<0.01.

[0126] As a result, as shown in FIG. 8, it was confirmed that among the ovariectomy group (OVX) the control group not treated with Agastache rugosa hot water extract (WEAR) had an increase in body weight compared to the Sham group, and it was confirmed that when WEAR was administered at 0.2 g/kg or 0.6 g/kg, respectively, weight gain due to ovariectomy was significantly inhibited.

[0127] In addition, as shown in FIG. 9, it was confirmed that the ovariectomy group (OVX) had reduced uterine weight due to uterine atrophy by ovariectomy compared to the Sham group, and administration of Agastache rugosa hot water extract (WEAR) did not affect the ovariectomy-induced uterine atrophy.

[0128] FIG. 10 a) is computed tomography 3D images of trabecular bone of the thigh bone in each experimental group; FIG. 10 b) is a graph showing bone volume (BV/TV, %); FIG. 10 c) is a graph showing trabecular bone thickness (Tb.Th, mm); and FIG. 10 d) is a graph showing trabecular bone count (Tb.N, 1/mm). Bone volume, trabecular bone thickness, and trabecular bone count were decreased in the ovariectomy (OVX) group compared to the Sham group, and administration of 0.2 g/kg and 0.6 g/kg of Agastache rugosa hot water extract (WEAR) significantly inhibited the decrease in bone volume, trabecular bone thickness, and trabecular bone count following ovariectomy (OVX).

INDUSTRIAL APPLICABILITY

[0129] The Agastache rugosa extract or the isoagastachoside or crude polysaccharide fraction included in the extract according to the present invention can be prepared based on plants being domestically cultivated, and thus is advantageous in terms of time and cost, and can effectively inhibit the differentiation, growth, and bone resorption functions of osteoclast progenitor cells, thus increasing bone density and the like, and accordingly, can be effectively used for the prevention, amelioration, or treatment of diseases related to bone loss caused by differentiation into osteoclasts or excessive activity of osteoclasts. In addition, the active ingredient, which is a plant-derived natural substance, is safe due to fewer side effects on the human body compared to chemical drugs, and has the effect of inhibiting osteoclast progenitor cells independently of estrogen, and thus can be widely used in osteoporosis occurring due to various causes other than menopause, so it has great industrial applicability.